本文關(guān)鍵詞： 貽貝粘蛋白 mgfp-基因 轉(zhuǎn)基因煙草 愈傷培養(yǎng)　出處：《基因組學(xué)與應(yīng)用生物學(xué)》2017年05期 　論文類型：期刊論文

【摘要】：海洋軟體動物貽貝(Mytilidae)足絲腺分泌的貽貝粘蛋白(mussel adhesive protein,MAP)即貽貝足絲蛋白(mussel foot protein,Mfp),不僅有水中高粘性及耐腐蝕性,而且無毒、無免疫原性,生物相容性好,其中Mfp-5粘性最強。現(xiàn)用的貽貝粘蛋白主要是天然獲取,但是其含量低易固化難獲得,基因工程重組貽貝粘蛋白的研究剛剛開始。前期我們在煙草中表達了地中海貽貝蛋白(mytilus galloprovincialis foot protein type 5,Mgfp-5)基因,得到轉(zhuǎn)mgfp-5基因煙草種子。本實驗將轉(zhuǎn)基因T1代種子無菌苗進行愈傷組織誘導(dǎo),在附加不同濃度的2,4-D、6-BA、NAA的MS培養(yǎng)基上,誘導(dǎo)率≥90.0%,其中2,4-D 1.0 mg/L、6-BA 0.5 mg/L、NAA 0.1 mg/L時誘導(dǎo)率為100%;愈傷組織固/液培養(yǎng)過程中均以"S"型曲線生長,固體培養(yǎng)時12~27 d鮮質(zhì)量增長率為3.73%,液體培養(yǎng)時間縮短6 d,在6~15 d時間內(nèi)鮮質(zhì)量會增加8.56~8.63倍;固/液培養(yǎng)的繼代愈傷組織,PCR、RT-PCR及Western blotting分別檢測到預(yù)期大小的電泳條帶,顯示了外源mgfp-5基因及Mgfp-5蛋白穩(wěn)定表達。穩(wěn)定表達Mgfp-5蛋白的愈傷組織,可以為獲得重組蛋白Mgfp-5的原材料提供新途徑,為研究植物表達貽貝粘蛋白提供參考。
[Abstract]:The mussel adhesive adhesive secreted by the foot silk gland of the marine mollusk Mytilida (Mytilida), which is called mussel foot protein, not only has high viscosity and corrosion resistance in water, but also has no toxicity, no immunogenicity, and good biocompatibility. The viscosity of Mfp-5 is the strongest. The mussel mucin in use is mainly obtained naturally, but its content is easy to solidify and difficult to obtain. Genetic engineering of recombinant mussel mucin has just begun. Previously, we expressed the MMP gene of mytilus galloprovincialis foot protein type 5 in tobacco. Mgfp-5 transgenic tobacco seeds were obtained. In this experiment, transgenic T1 seeds were induced by callus induction on MS medium supplemented with different concentrations of 2H4 DX 6-BANAA. The induction rate was more than 90.0. The inductive rate of callus was 100 at 1.0 mg / L 1.0 mg / L 6-BA 0.5 mg / L NAA 0.1 mg/L, and the callus grew on a "S" curve during solid / liquid culture, and the induction rate was more than 90% at 0. 1 mg 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). The growth rate of fresh mass was 3.73, the time of liquid culture was shortened by 6 days, and the fresh mass increased by 8.56 ~ 8.63 times in 6 ~ 15 days after solid / liquid culture, and the electrophoresis bands of expected size were detected by RT-PCR and Western blotting in subculture callus of solid / liquid culture, respectively. The stable expression of exogenous mgfp-5 gene and Mgfp-5 protein and the stable expression of Mgfp-5 protein in callus could provide a new way to obtain the raw material of recombinant protein Mgfp-5 and provide a reference for studying the expression of mussel mucin in plants.
【作者單位】： 西北大學(xué)生命科學(xué)學(xué)院/陜西省生物技術(shù)重點實驗室/西部資源與現(xiàn)代生物技術(shù)省部共建教育部重點實驗室;
【基金】：國家自然科學(xué)基金(31270411;31572665) 陜西省科技廳社會發(fā)展攻關(guān)計劃(2012K12-01-02) 陜西省教育廳專項資金(12JK0827);陜西省教育廳重點實驗室項目(12JS105)共同資助
【分類號】：Q943.1
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本文編號：1526968